Liver disease affects more than 500 million people worldwide. Organ transplantation is the gold standard for treatment of liver failure, but organ shortages are acute. Cell-based therapies, such as cell transplantation, engineered hepatocellular tissue constructs and bioartificial liver devices, have long held promise as alternatives to whole organ transplantation. Such therapies require the use of human hepatocytes due to substantial species-specific differences between animal and human hepatocellular functions.
Human hepatocytes, within the stromal context of the liver in vivo, are capable of extensive proliferation, enough to fully regenerate the liver following up to 66% partial hepatectomy. However, this ability is lost in vitro, despite extensive gene-by-gene and protein-by-protein approaches towards harnessing this tremendous replication potential of human hepatocytes. Investigations have yielded many different culture conditions that can support moderate expansion of mouse and rat hepatocytes, including a multi-factor media formulation that expands rat hepatocytes through a dedifferentiated bi-potential intermediate. However, translation of these findings to human cultures has not been reported. Human cells are critical for cell-based therapies due to substantial differences between animal and human hepatocellular functions, including apolipoprotein expression, metabolic regulation of cholesterol, and phase I detoxification enzymes. To overcome the growth limitations of primary human cells, various human hepatocyte cell lines have been developed. Although these cell lines are growth-competent, they introduce safety concerns and underperform primary cells in terms of liver function.
In addition to their use in the transplantation therapies to treat liver diseases, human hepatocytes are in high demand for drug toxicity screening and development, because of their critical function in the detoxification of drugs or other xenobiotics, as well as endogenous substrates. However, human primary hepatocytes quickly lose their functions when cultured in vitro. Moreover, the drug metabolic ability of human primary hepatocytes exhibits significant variance among individuals. The availability of an unlimited supply of patient-specific functional hepatocytes would greatly facilitate both the drug development and the eventual clinical application of hepatocyte transplantation.
Alternative sources for hepatic cells are being investigated, such as various stem cell populations. Stem cells hold great promise as a biologics source due to their ability to self-renew without limit and to differentiate along many lineages. Induced pluripotent stem cells (iPS cells) additionally create the possibility of establishing patient-specific cell types, thus empowering personalized medicine. Human iPS cells are generated from somatic cells via forced expression of reprogramming factors, and can be differentiated towards hepatocyte-like cells (iHeps) in a step-wise manner, using defined factors. Mouse iHeps can also be generated directly from fibroblasts via cellular reprogramming. Although diverse stem and progenitor cell types exhibit vast potential for integration into hepatic treatments, many challenges remain, including the ability to completely dictate differentiation into fully mature hepatocytes. While human iHeps closely resemble mature hepatocytes, key differences in their phenotypes tlimit their use as a renewable source of functional hepatocytes. Notably, iHeps persistently express fetal markers like alpha fetoprotein (AFP) and lack key mature hepatocyte functions, as reflected by drastically reduced activity (0.1%) of many CYP450s (e.g. CYP2A6 and CYP3A4). Consequently, for decades, human hepatocyte sourcing has been a bottleneck for many fields of research and clinical therapies.
There is a need for identifying compounds that induce proliferation and/or differentiation of cells, such as somatic cells. Such compounds may be used, for example, to produce hepatic lineage cells for therapeutic and research use, such as human hepatocytes. The present invention addresses and satisfies this unmet need.